The use of reporter molecules or labels to qualitatively or quantitatively monitor molecular events is well established in assays used for medical diagnosis, for the detection of toxins and other substances in industrial environments, and for basic and applied research in biology, biomedicine, and biochemistry. Such assays include immunoassays, nucleic acid probe hybridization assays, and assays wherein production of a reporter molecule is due to transcription from a particular promoter. Reporter molecules or labels in such assay systems have included radioactive isotopes, fluorescent agents, enzymes and chemiluminescent agents.
Among the assay systems, in which chemiluminescence has been employed to monitor or measure events of interest, are those in which the activity of a bioluminescent enzyme, a luciferase, is measured. Use of luciferase assays, however, has not been widespread because of the brevity and pattern of the light emission in the assays. With beetle luciferases, this emission involves very rapid attainment of a peak intensity, i.e., a flash of light, followed by a slower, but still problematically rapid, decay to an even more slowly decaying "steady-state" intensity of, initially, approximately only about 10% of the peak intensity. The brevity of the intense light emission usually requires specialized laboratory procedures, such as rapid injection of the enzyme into a substrate solution, to prepare the bioluminescent reaction mixture in carrying out the assay. The need to measure the light emitted during the "flash" continues as a cause of experimental problems. It remains difficult, even with present day luminometers, to precisely measure such light.
Light-emitting systems have been known and isolated from many luminescent organisms, including certain bacteria, protozoa, coelenterates, molluscs, fish, millipedes, flies, fungi, worms, crustaceans, and beetles, particularly the fireflies of the genera Photinus, Photuris, and Luciola and click beetles of genus Pyrophorus. In many of these organisms, enzymatically catalyzed oxidoreductions take place in which the free energy change is utilized to excite a molecule to a high energy state. Then, when the excited molecule spontaneously returns to the ground state, visible light is emitted. This emitted light is called "bioluminescence."
Beetle luciferases, particularly that from the firefly species, Photinus pyralis, have served as paradigms for understanding of bioluminescence since the earliest studies. The P. pyralis luciferase is an enzyme which appears to have no prosthetic groups or tightly bound metal ions and has 550 amino acids and a molecular weight of about 60,000 daltons; the enzyme has been available to the art in crystalline form for many years. Studies of the molecular components in the mechanism of firefly luciferases in producing bioluminescence have shown that the substrate of the enzymes is firefly luciferin, a polyheterocyclic organic acid, D-(-)-2-(6'-hydroxy-2'-benzothiazolyl)-.DELTA..sup.2 -thiazolin-4-carboxylic acid (hereinafter referred to as "luciferin", unless otherwise indicated).
The beetle luciferase-catalyzed reaction which yields bioluminescence (hereinafter referred to simply as "the beetle luciferase-luciferin reaction") has been described as a two-step process involving firefly luciferin, adenosine triphosphate (ATP), and molecular oxygen. In the initial reaction, the luciferin and ATP react to form luciferyl adenylate with the elimination of inorganic pyrophosphate as indicated in the following reaction: EQU E+LH.sub.2 +ATP+Mg.sup.2 .fwdarw.E.multidot.LH.sub.2 -AMP+PP.sub.i
where E is the luciferase, LH.sub.2 is the luciferin, Mg.sup.2+ is magnesium ion, and PP.sub.i is pyrophosphate. The luciferyl adenylate, LH.sub.2 -AMP, remains tightly bound to the catalytic site of luciferase. When this form of the enzyme is exposed to molecular oxygen, the enzyme-bound luciferyl adenylate is oxidized to yield oxyluciferin (L.dbd.O) in an electronically excited state. The excited oxidized luciferin emits light on returning to the ground state as indicated in the following reaction: ##EQU1## One quantum of light is emitted for each molecule of luciferin oxidized. The electronically excited state of the oxidized luciferin is a state that is characteristic of the luciferase-luciferin reaction of a beetle luciferase; the color (and, therefore, the energy) of the light emitted upon return of the oxidized luciferin to the ground state is determined by the enzyme, since different species of beetles having the same luciferin emit light of different colors.
When light emission is initiated by injection of ATP into a reaction mixture containing luciferase, Mg.sup.2+, and luciferin, where all components are near or at saturating concentrations, one observes a rapid increase in intensity followed by a rapid decrease in the first few seconds, followed by a further decay that may last hours. This decrease in the rate of reaction has been thought to be due to product inhibition.
Luciferase has been used as a means of assaying minute concentrations of ATP; as little as 10.sup.-16 molar ATP can be detected with high quality preparations of the enzyme. The luciferase-luciferin reaction is highly specific for ATP. For example, deoxy-ATP produces less than 2% of the light generated by ATP, and other nucleoside triphosphates produce less than 0.1%.
Coupling the concentration of ATP with the activity of other enzymes has allowed luciferase to become a biochemical reporter molecule for these other enzymes as well as other compounds.
The availability of beetle luciferases for use as reporters in other assays is not a problem. Such uses have been limited, however, by the problematic kinetics of light emission in the luciferase-luciferin reaction. But for such problematic kinetics, readily available beetle luciferases could have been employed in applications, such as immunoassays, such as enzyme-linked immunosorbent assays, in which an enzyme serves as reporter, and nucleic acid probe hybridization assays, in which an enzyme serves as a reporter.
Beyond the availability of crystalline luciferases isolated directly from the light organs of beetles, CDNAS encoding luciferases of several beetle species (including, among others, the luciferase of P. pyralis(firefly) , the four luciferase isozymes of P. plagiophthalamus(click beetle), the luciferase of L. cruciata(firefly) and the luciferase of L. lateralis)(de Wet et al., Molec. Cell. Biol. 7, 725-737 (1987); Masuda et al., Gene 77, 265-270 (1989); Wood et al., Science 244, 700-702 (1989); European Patent Application Publication No. 0 353 464) are available. Further, the CDNAS encoding luciferases of any other beetle species, which make luciferases, are readily obtainable by the skilled using known techniques (de Wet et al. Meth.Enzymol. 133, 3-14 (1986); Wood et al., Science 244, 700-702 (1989). With the cDNA encoding a beetle luciferase in hand, it is entirely straightforward for the skilled to prepare large amounts of the luciferase in highly pure form by isolation from bacteria (e.g., E. coli), yeast, mammalian cells in culture, or the like, which have been transformed to express the CDNA. Various cell-free systems, that have recently become available to make proteins from nucleic acids encoding them, can also be used to make beetle luciferases.
Further, the availability of CDNAS encoding beetle luciferases and the ability to rapidly screen for CDNAS that encode enzymes which catalyze the luciferase-luciferin reaction (see de Wet et al., Meth. Enz., supra, and Wood et al., supra) also allow the skilled to prepare, and obtain in large amounts in pure form, mutant luciferases that retain activity in catalyzing production of bioluminescence through the luciferase-luciferin reaction. Such a mutant luciferase will have an amino acid sequence that differs from the sequence of a naturally occurring beetle luciferase at one or more positions. In the present disclosure, the term "beetle luciferase" comprehends not only the luciferases that occur naturally in beetles but also the mutants, which retain activity in providing bioluminescence by catalyzing the luciferase-luciferin reaction, of such naturally occurring luciferases.
The ready availability of CDNAS encoding beetle luciferases makes possible the use of the luciferases as reporters in assays employed to signal, monitor or measure genetic events associated with transcription and translation, by coupling expression of such a CDNA, and consequently production of the enzyme, to such genetic events.
Thus, while the potential uses for beetle luciferases as reporter molecules have become increasingly important and quite varied, the brevity and pattern of the light emission caused by the enzymes has limited their utility in practice. It would be desirable to enhance the utility of beetle luciferases as reporters by effecting with them more efficient light production, i.e., light emission at a more nearly continuous, yet high, rate.
One approach, which achieved some popularity, to solving the problem of the kinetics of the luciferase-luciferin reaction and the associated difficulty of precisely measuring light emitted during the flash, was to use various inhibitors of the enzyme, which were reported to prevent the flash from occurring or to prolong light production. One such agent is arsenate. Arsenate lowers flash height and tends to prolong the light emission for a given amount of ATP but reduces sensitivity for detecting ATP. While luciferase preparations containing arsenate remained commercially available until recently, use of such preparations is no longer favored. In part this is because the need for such use can be avoided in some applications with the use of sophisticated light-measuring instrumentation.
However, even with such sophisticated instrumentation, specialized laboratory procedures, such as an injection format for rapidly mixing the enzyme and substrate, are still required. Improving the kinetics of light production for the enzymatic reaction, to avoid the need for such specialized and cumbersome procedures, would greatly expand the utility of luciferases as reporters.
A number of compounds, besides arsenate salts, has also been reported to affect the pattern of light production from the beetle luciferase-luciferin reaction. Phosphate salts were employed for the same purpose as the arsenate salts but were not favored because the required presence of magnesium ion in the assay systems led to the undesirable precipitation of magnesium phosphate when phosphate salts were used.
The cofactor, coenzyme A (CoA), has been reported to affect the pattern of light emission in the luciferin-luciferase reaction. Airth et al., Biochimica et Biophysica Acta, vol. 27 (1958) pp. 519-532, report that, when CoA is added to a firefly luciferin-firefly luciferase reaction mixture, there is no effect on the initial peak of light intensity but luminescence will continue at a higher level for a time period that is proportional to the total CoA added. Airth et al. have shown that the total light emitted is greater in the presence of CoA than in its absence.
Airth et al. also report that cysteine and glutathione do not stimulate light emission and, without providing details, that hydroxylamine stimulates and thioethanolamine slightly stimulates emission in a manner similar to CoA.
The teaching of Airth et al. concerning the effect of CoA on light emission from the beetle luciferase-catalyzed reaction of luciferin, ATP and oxygen, is suspect. Subsequent to the Airth et al. report, the effect of CoA on luciferase was explained on the basis of prevention by CoA of inhibition of the enzyme by dehydroluciferin, a compound thought to be a significant contaminant of the luciferin used by Airth et al. and subsequent workers, who purified the luciferin from fireflies. Luciferin employed in more recent times, and today, is prepared synthetically and, as such, is substantially free of dehydroluciferin. Synthetic preparations of luciferin typically are contaminated with less than 1%, and preferably less than 0.3%, dehydroluciferin by weight relative to luciferin. Thus, CoA would be expected to have no effect on luciferase activity with synthetically prepared luciferin. The teaching of Airth et al. and subsequent workers on stimulation of light emission with beetle luciferases by CoA (and other compounds mentioned in the Airth et al. reference) has been completely ignored for more than 30 years in efforts to broaden the practical applicability of assays based on luciferase-catalyzed light emission. For example, CoA has never been suggested as a substitute for arsenate, notwithstanding the recognized undesirability of arsenate.
It has also been reported that other sulfhydryl compounds contribute to the stability of luciferases during preparation and storage of the enzymes. U.S. Pat. No. 4,833,075 discloses that dithiothreitol (DTT) will maintain luciferase activity at a level of 50% in an aged Photinus pyralis luciferase solution which, without the DTT, would have only 10% residual enzymatic activity compared to a freshly prepared luciferase solution. U.S. Pat. No. 4,614,712 describes that, when bacterial luciferase has been inactivated by disulfide formation, enzyme activity may be restored by addition of DTT, .beta.-mercaptoethanol (.beta.-ME), or other reducing agents. Although beetle luciferases and bacterial luciferases differ in structure and action, both appear to have a reactive sulfhydryl group which may be protected from general oxidation by certain reducing agents.
However, it has been thought in the art that dithiothreitol (DTT) and similar thiol reagents, at concentrations above about 5 mM, would inhibit light emission catalyzed by luciferases.
Despite recognition and study of various aspects of the chemistry of luciferases, the prior art has provided little in the way of practical techniques for more efficient light production from the luciferase-luciferin reaction to increase the utility of the resulting luminescence as a detection mode.